DE1598739A1 - Means for determining the cholinesterase activity in serum - Google Patents

Description

B. Merck 1 π QQT OQ

Aktiengesellschaft I 030 / ÖS ₂ . _July 1969

Darmstadt

Means for determining the cholinesterase activity

in the serum

The determination of the activity of serum cholinesterase is necessary for the early detection of certain diseases is extremely important. For example, decreased cholinesterase activity on degenerative liver diseases or on poisoning with organic phosphoric acid esters inferred and the degree poisoning can be determined. Furthermore, the effectiveness of anticholinesterases ^. those at different Diseases administered are determined. It is also very important, among other things, before using muscle relaxants To determine the level of cholinesterase in the patient's serum during surgery.

Various methods of determination have already been found in the literature known of serum cholinesterase activity. For example, it has been proposed to test a serum sample with a low Combine the amount of substrate (acetylcholine) in the presence of a color indicator in solution or on a strip of paper and the The color change caused by the acetic acid released from the substrate should be estimated after a certain period of time or to measure colorimetrically. The previously known test equipment and test methods, however, have significant shortcomings. The exam using this method is tedious, since the outlay on equipment is great and, in particular, the incubation time is long. Further The previously known paper strip methods provide largely imprecise results that only provide rough information about the Allow oholinesterase content.

■ It has now been found that the difficulties mentioned do not occur when a carrier is used for the determination of the serum cholinesterase activity that contains phenol red, naphtholphthalein

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and substrate is occupied.

The present invention is therefore a means for determining the cholinesterase activity in serum, which consists of a Absorbent carrier consists of the substrate and as an indicator phenolic, red and contains naphtholphthalein.

With the agent according to the invention, the cholinesterase content can be determined in a simple manner without a great deal of resources will. It is particularly advantageous that only short incubation times are required when using the new agent. That's the way it is possible, using the carrier covered with phenol red, with naphtholphthalein and substrate according to the invention, the choline ester ' The ase content should be indicated semi-quantitatively after 6 to 8 minutes, for example. The incubation times are therefore noticeably shorter than when using of known means with comparable equipment. Compared to some previously used means for determining jig of the cholinesterase activity, the new carrier according to the invention is distinguished by the fact that not several times at certain lid intervals, but only once after a short time the color of the indicator has to be read off. The agent according to the present invention thus provides a considerable saving in labor in the determination of the cholinesterase activity in comparison to the known agents Serum, which is particularly effective when performing simultaneous determinations.

Any absorbent is used as a carrier for the agent according to the invention suitable organic or inorganic material, preferably layered cellulose carriers, e.g. filter paper, furthermore Carrier made of glass fibers, silica gel or rods made of suitable material such as aluminum oxide.

The indicator and the acetylcholine used as a substrate are applied in a suitable form, advantageously in the form of a solution Applied carrier, the solvent used is then allowed to evaporate. Suitable solvents for both

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the substrate as well as for the indicator are, if appropriate, organic solvents mixed with water, such as alcohol, in particular lower alcohols, e.g. methanol, ethanol, isopropanol, also ketones and ethers such as acetone, dioxane, tetrahydrofuran. That The substrate can be applied to the carrier in the form of a solution of the free acetylcholine. However, it has proven to be special expediently highlighted »the carrier with a solution of a salt of acetylcholine, preferably with a solution of a hydrohalide, especially the hydrochloride or with the solution of the hydrobromide or sulfate or a mixture of the salts soak.

According to the invention, a mixture of phenol red with naphtholphthalein is used as an indicator. The applied indicator mix is particularly favorable »because even in the weakly alkaline range there are particularly clear changes in parba with a slight change in the pH and is therefore also very suitable for measuring the oholinesterase content of older sera. The indicator solution should a pH-Y / ert of about 5 to about 8.5 i, preferably pH 6, have If necessary, the solution of the indicator and substrate must be adjusted to the desired pH before application to the carrier. When using acidic salts of acetylcholine, this can be done by adding the appropriate amount of an alkaline one. Substance, preferably an alkaline hydroxide such as an alkali metal hydroxide, especially sodium hydroxide or potassium hydroxide or also another alkaline compound, e.g. an alkaline salt such as an alkali carbonate or bicarbonate, e.g. sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate · reached v / earth. If you use the basic, reactive free acetylcholine, the desired pH can be adjusted by adding acidic substances, e.g. an acid such as hydrochloric acid will. The alkaline or acidic substance required to adjust the pH is expediently in the form of its diluted, for example added to aqueous or alcoholic solution,

It has been found to be beneficial, indicator, substrate and the alkaline or acidic substance that may be required to be applied in the form of a common solution to the carrier.

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It is advantageous, for example, to combine the components in an alcoholic solution in which-about 5-30 $, in particular about 15 ^c / ° acetylcholine and / or acetylcholine salts and about 0.05-0.2 ^, in particular about 0.1 5 ^ Indicator dye is included, use. The carrier can also be covered with substrate and indicator or their solutions one after the other. The order in which the constituents are added to a common solvent or the order in which they are applied to the carrier is arbitrary. The carrier material impregnated in this way, which may for example be in the form of strips, plates or in the form of other shaped bodies, is expediently brought to a suitable format after the solvent has evaporated. For example, a filter paper impregnated in the manner described is cut into squares or rectangles or strips. When storing the impregnated carrier, exposure to light and air should be excluded as far as possible in order to avoid a change in color.

The application of the new agent is extremely easy. The impregnated carrier, e.g. the test paper, has a defined Amount of the serum to be tested, e.g. 0.05 ml / cm test paper, at temperatures between 15 and 25 ° C, preferably at 20 0, brought together. After a set time (between about and 10 minutes, preferably after 6-8 minutes), the the acetic acid released caused the carrier to hue with the colors of a color scale calibrated under the same conditions compared. In this way, the concentration of cholinesterase can be read off directly. In special cases it can be useful be able to choose a slightly longer incubation time, e.g. 15 minutes «

The following tables show the color and pH values with the associated cholinesterase concentrations when tested 0.04 ml

'2
Serum entered per cm of carrier after an incubation time of 5 or 10 minutes at a temperature between 15 and 25 ^° C.

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color of the carrier PH value Cholinesterase content of the serum diagnostic assessment ^ IU / ml very much humiliated dark purple 8.0 0 very much humiliated Intermediate value 1 7.8 0.6 severely humiliated Intermediate value 2 7.6 1.3 humiliated brick red 7.4 2.0 normal Intermediate value 7.2 2.8 normal Red orange 7.0 3.6 elevated Intermediate value 6.8 4.4 fetal increased yellow-orange 6.6 5.2 ' very much increased Intermediate value 6.4 6.0 very much increased dark yellow 6.2 6.8

Calibration table 1: Cholinesterase content and color of the phenol red /

üaphtholphthalein and acetylcholine coated carrier

ρ when testing 0.04 ml of serum per cm of carrier

15-25 ⁰ C and an incubation time of 5 minutes.

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• dark purple - 6 pH value - 1598739. diagnostic assessment Color of the carrier Intermediate value 1 Cholinesterase content of the serum very much humiliated Intermediate value 2 8.0 IU / ml very much humiliated brick red 7.8 0 very much humiliated Intermediate value 7.6 0.6 severely humiliated . * ·
Red orange 7.4 1.0 severely humiliated [I intermediate value
II yellow-orange 7.2 1.4 humiliated Intermediate value 7.0 1.8 normal dark yellow 6.8 2.2 normal Intermediate value . 6.6 2.6 normal lemon yellow 6.4 3.0 elevated 6.2 3.5 elevated 6.0 4.0 greatly increased 5.8 4.5 5.0

Calibration table 2; Cholinesterase content and color of the phenol red /

Naphtholphthalein and acetylcholine coated carrier

ρ when testing 0.04 ml of serum per cm of carrier

15 - 25 ⁰ C and an incubation time of 10 minutes

. It is particularly advantageous to carry out several determinations side by side with the means of the invention. Such simultaneous determinations can for example be carried out on a pallet made of suitable material such as metal, porcelain, glass or a plastic after applying the serum samples and the impregnated carrier in the wells of the pallet, it is expedient a transparent cover plate, e.g. made of glass or plexiglass, is used. The paper can also be applied to an object

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slides, moistened with serum and covered with a second slide. After a short time, e.g. 6 to 8 Minutes, then the cholinesterase content for the whole Test series can be read off by comparing the color shades achieved with the colors in the calibration table.

example 1

62 mg phenol red, 62 mg naphtholphthalein and 19 g acetylcholine hydrochloride are dissolved in 100 ml of a mixture of acetone and water (ratio of solvent components 10: 6). By A potassium hydroxide solution is added to adjust the pH to 6.0. With the indicator solution obtained in this way, glass fiber paper, filter paper or filter cardboard are impregnated by briefly immersing them.

Example 2

70 mg phenol red, 70 mg naphtholphthalein and 15 g aoetylcholine hydrochloride are dissolved in 100 ml of methanol. The solution is then adjusted to a pH of 6.0 with n / 10 potassium hydroxide solution The indicator solution prepared in this way becomes absorbent carriers impregnated analogously to Example 3.

Example 3

150 mg phenol red, 150 mg haphtholphthalein uni. 25 g acetylcholine hydrochloride are dissolved in 100 ml of 70% ethanol. Afterward the solution is brought to a pH of 7 »ö by adding sodium hydroxide solution set. With the indicator solution prepared in this way, ready-to-use Carrier impregnated as indicated in Example 3.

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Example 4

100 mg plienol red, 100 mg naphtholphthalein and 15 g acetylcholine hydrochloride are dissolved in 100 ml of methanol and then adjusted to pH 6.0 by adding sodium hydroxide solution. Sheets of filter paper are impregnated with the solution obtained. After the solution has dried, the filter paper is in Applied in the form of strips to a plastic film and sealed with a second film. Then the bow is with the sealed, impregnated filter paper perpendicular to the applied paper strip in the usual way in strips cut.

In a variation of the manufacturing process described above the filter paper sheet is first applied to the plastic film and only then soaked with the solution.

The test strips made of plastic film with applied indicator paper are then determined for cholinesterase used.

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Claims (1)

Claim. 1 · Means for determining the cholinesterase activity im Serum, consisting of an absorbent carrier containing a color indicator and substrate, characterized in that, that as an indicator phenol red together with naphtholphthalein, is used. BAD OR * GlNAt 0Q9882 / 0A21